Using amines or amino acids as mobile phase modifiers in chromatography

ABSTRACT

This invention relates to the use of amine, amino acid and amino acid ester mobile modifiers in normal phase chromatography to improve the resolution and or productivity of peptide and lipopeptide purification. This chromatographic method can be sued for either analytical or preparative scale purification.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage of International ApplicationNo. PCT/US03/33978 filed on Oct. 24, 2003, which claims priority under35 USC 119(e) from U.S. Provisional Application No. 60/422,356 filed onOct. 30, 2002.

FIELD OF THE INVENTION

This invention relates to the use of amines, amino acids or amino acidesters as mobile phase modifiers in normal phase chromatography ofpeptide and lipopeptide compounds.

BACKGROUND OF THE INVENTION

In the past, the resolution of key impurities such as Pneumocandins B₅and E₀ from Pneumocandin B₀ in the silica gel HPLC purification waspoor. Some analogs were only partially resolved from the main productpeak under preparative conditions. To achieve the desired productpurity, the limited resolution required that the purification step berun at low column loading, which limited productivity.

Lipopeptides, such as Pneumocandin B₀, are often the product of afermentation process. During such a fermentation process, many veryclosely related analogues are produced along with the desired product.Normal phase chromatography systems are frequently used to purify thecrude fermentation product. A normal phase chromatography system usuallyconsists of a stationary phase and a mobile phase. For purification of apeptide or lipopeptide, the stationary phase can be silica gel oralumina, and the mobile phase can be a single solvent or a mixture ofsolvents, which includes organic solvents and water.

Silica gel chromatography and other types of normal phase chromatographyare useful for separating these analogues. However, in practice, theresolution of certain close analogues from the desired product is oftenpoor and not satisfactory, because the resolution is not great and oftenthere is overlap. To achieve the desired purity of the main product at areasonable yield requires restricting the amount of material (oftenreferred to as feed or column load) loaded onto the column per run,which limits the productivity of the operation.

The purification of Pneumocandin B₀ falls into this category. Thechromatography utilizes a mobile phase consisting of a mixture ofsolvents, specifically ethyl acetate (EtOAc), methanol (MeOH) and water,on a silica gel column. Pneumocandin B₀, with a molecular weight of 1065Daltons, is a natural product and serves as an intermediate in theproduction of Caspofungin acetate (Cancidas®). Pneumocandin B₀ isproduced as a secondary metabolite by fermentation of the fungus Glarealozoyensis. See U.S. Pat. Nos. 5,194,377 and 5,202,309. The structuresof Pneumocandin B₀ and two of the key analog impurities, all comprisedof a cyclic hexapeptide coupled with dimethylmyristate side chain, areshown in Formula I and Table 1.

TABLE 1 Pneumocandin B₀ and two of its analogs Compound R¹ R² R³ R⁴ R⁵R⁶ Pneumocandin B₀ OH OH Me OH H OH Pneumocandin B₅ OH H Me OH H OHPneumocandin E₀ OH OH Me OH H H

Silica gel chromatography exploits the subtle variations in bindingaffinity of the hydroxy-rich cyclic hexapeptide core of the desiredproduct and the analog impurities, including Pneumocandins B₅ and E₀, toeffect a separation. In the silica gel HPLC purification, PneumocandinsB₅ and E₀, two of the key analog impurities co-produced in thefermentation of Pneumocandin B₀, elute very close to Pneumocandin B₀.Therefore, to meet the target impurity levels in the purified materialfor these and similar analog compounds, the quantity of crudePneumocandin B₀ that can be loaded onto the column is limited. As aresult, significant efforts have been made to improve the resolution ofkey impurities. For instance, the ternary ethyl acetate-methanol-watermobile phase has been balanced to optimize resolution betweenPneumocandin B₀ and key analog impurities. D. J. Roush, F. D. Antia, K.E. Göklen J. Chromatography A, 827 (1998) 373-389.

SUMMARY OF THE INVENTION

This invention discloses the use of mobile phase modifiers, includingamino acids, amino acid esters or amines, during the HPLC purificationof a lipopeptide or peptide. The mobile phase modifiers bind to thestationary phase and modify the binding characteristics of thestationary phase improving the resolution and or purification of desiredlipopeptide or peptide from its related impurities.

This invention is useful in analytical chromatography and even morevaluable for preparative chromatography (i.e., using chromatography as alarge-scale purification technique). Specifically, amino acids, aminoacid esters or amines can be used as mobile phase modifiers in thechromatographic purification of peptides or lipopeptides, for examplethe purification of Pneumocandin B₀, which is the natural productstarting material (a fermentation product) used to prepare Caspofunginacetate (Cancidas®). The invention would also be useful in purifying thefermentation product precursor for other lipopeptides, such asMicafungin, Anidulafungin, Cilofungin and Daptomycin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B.

Preparative HPLC chromatogram of silica gel chromatography of crudePneumocandin B₀, where the column feed contains a small amount ofproline. FIG. 1A. First run on the column. FIG. 1B. Eighteenth run onthe column. Improved resolution of analogue impurities is seen in thelater run due to the adsorption of proline from the feed solution ontothe column.

FIGS. 2A, 2B and 2C

FIG. 2A. Plot of retention time of Pneumocandin B₀ after injectingproline-containing solution onto the column, showing increased retentiontime, which returns to the original retention time, as the proline isslowly desorbed from the column. FIGS. 2B and 2C. Chromatogram of atypical run just before (2B) and after (2C) injecting proline onto thecolumn. (analytical scale 5 μ YMC silica column using 87/9/7 v/v/v ethylacetate/methanol/water).

FIGS. 3A, 3B and 3C

Silica gel HPLC chromatograms for Pneumocandin B₀ crude eluted withmobile phase: (3A) without L-proline, (3B) with 0.26 mM L-proline added,and (3C) with 0.65 mM L-proline added. Demonstrates increased retentiontime of Pneumocandin B₀ and increased resolution from analog impuritieswith increasing levels of proline added. (Pneumocandin B₀ retention timeis 22 minutes with 0.65 mM L-proline).

FIG. 4

Silica gel BPLC chromatogram for Pneumocandin B₀ crude eluted using 20%ethyl acetate and 80% 87/9/7 v/v/v ethyl acetate/methanol/water as themobile phase. This mobile phase mixture results in increased retentiontime of Pneumocandin B₀, but without the improved resolution from itsanalogs observed when proline is added to the mobile phase (see FIG. 3).(Pneumocandin B₀ retention time is 23 minutes).

FIGS. 5A through 11B

Silica gel HPLC chromatograms for Pneumocandin B₀ crude eluted (A)without mobile phase modifier (control) and (B) with the addition ofvarious amino acids to the mobile phase. Demonstrates that addition ofall the amino acids have an affect on the Pneumocandin B₀ retentionand/or its resolution from its analogs. (analytical scale 5 μ YMC silicacolumn using 87/9/7 v/v/v ethyl acetate/methanol/water).

FIGS. 5A and 5B

FIG. 5A. Control prior to exposure of column totrans-4-hydroxyl-L-proline.

FIG. 5B. Chromatogram after exposure of column to mobile phase modifiedwith trans-4-hydroxyl-L-proline, showing similar retention time ofPneumocandin B₀ to the control, but different resolution from its analogimpurities.

FIGS. 6A and 6B.

FIG. 6A. Control prior to exposure of column to L-valine.

FIG. 6B. Chromatogram after exposure of the column to mobile phasemodified with L-valine, showing changes from the control in both theretention time of Pneumocandin B₀ and the resolution from its analogimpurities.

FIGS. 7A and 7B

FIG. 7A. Control prior to exposure of column to L-lysine.

FIG. 7B. Chromatogram after exposure of the column to mobile phasemodified with L-lysine, showing changes from the control in both theretention time of Pneumocandin B₀ and the resolution from its analogimpurities.

FIGS. 8A and 8B

FIG. 8A. Control prior to exposure of column to L-methionine.

FIG. 8B. Chromatogram after exposure of the column to mobile phasemodified with L-methionine, showing changes from the control in both theretention time of Pneumocandin B₀ and the resolution from its analogimpurities.

FIGS. 9A and 9B.

FIG. 9A. Control prior to exposure of column to D-proline.

FIG. 9B. Chromatogram after exposure of the column to mobile phasemodified with D-proline, showing changes from the control in both theretention time of Pneumocandin B₀ and the resolution from its analogimpurities.

FIGS. 10A and 10B

FIG. 10A. Control prior to exposure of column to L-threonine.

FIG. 10B. Chromatogram after exposure of the column to mobile phasemodified with L-threonine, showing changes from the control in both theretention time of Pneumocandin B₀ and the resolution from its analogimpurities.

FIGS. 11A and 11B.

FIG. 11A. Control prior to exposure of column to glycine.

FIG. 11B. Chromatogram after exposure of the column to mobile phasemodified with glycine, showing changes from the control in both theretention time of Pneumocandin B₀ and the resolution from its analogimpurities.

FIGS. 12A and 12B

Silica gel HPLC chromatograms for Pneumocandin B₀ crude eluted (A)without (control) and (B) with the addition of diethylamine to themobile phase. Demonstrates that diethylamine addition has a very strongaffect on the Pneumocandin B₀ retention and its resolution from itsanalogs. (analytical scale 5μ YMC silica column using 87/9/7 v/v/v ethylacetate/methanol/water).

DETAILED DESCRIPTION OF THE INVENTION

A method for the purification of a peptide or a lipopeptide by using amobile phase modifier in a normal phase chromatography system to improvethe selectivity and/or productivity of the purification is disclosed.The method as recited above, wherein the mobile phase modifier isselected from a group consisting of an amine, an amino acid or an aminoacid ester. The method as recited above, wherein the normal phasechromatography system includes a mobile phase and a stationary phase.The method as recited above, wherein the stationary phase is selectedfrom silica gel and alumina. The method as recited above, wherein themobile phase is a solvent system comprising one or more solvents. Themethod as recited above, wherein the mobile phase modifier is an amine.The method as recited above, wherein the amine is a primary, secondary,or tertiary amines, including lower alkyl (C₁-C₆ alkyl) amines, lowerdialkyl (C₁-C₆ alkyl) amines and/or aromatic (C₆-C₁₀ aryl) amines.Examples of amines, which are useful in the method, as recited above aremethylamine, ethylamine, diisopropylamine, diethylamine, dimethylamine,ethylmethylamine, triethylamine, propylamine, aniline anddimethylaniline. The method as recited above, wherein the mobile phasemodifier is an amino acid or amino acid ester. The method as recitedabove, wherein the amino acid or amino acid ester mobile phase modifieris selected from the group consisting of: L-amino acids, D-amino acids,L-amino acid esters and D-amino acid esters. The method as recitedabove, wherein the amino acid or amino acid ester mobile phase modifieris selected from: L-proline, D-proline, trans-4-hydroxy-L-proline,trans4-hydroxy-D-proline, glycine, L-threonine, D-threonine, L-lysine,D-lysine, L-methionine, D-methionine, D-valine, L-valine and esters ofthe aforementioned band D-amino acids. The method as recited above,wherein the amino acid is selected from: L-proline and D-proline.

An embodiment of the invention is the method of purifying a peptide or alipopeptide by using an amine or amino acid mobile phase modifier in anormal phase chromatography system with a stationary phase of alumina orsilica gel to improve the resolution and/or productivity of thepurification, wherein the purification is of a peptide. Examples ofpeptides are linear and cyclic amino acid chains under 1800 molecularweight, formed by the combination of the amino group of one amino acidwith the carboxyl group of another in an amide bond, and produced assecondary metabolites of microorganisms. Other examples of peptides areoxytocin and bradykinin.

A further embodiment of the invention is the method of purifying apeptide or a lipopeptide by using an amine or amino acid mobile phasemodifier in a normal phase chromatography system with a stationary phaseof alumina or silica gel to improve the resolution and/or productivityof the purification, wherein the purification is of a lipopeptide. Themethod as recited above, wherein the lipopeptide is selected from afermentation product precursor of Caspofungin, Micafungin Andulifungin,Cilofungin, and Daptomycin. The method as recited above, wherein thelipopeptide is pneumocandin B₀. The method as recited above, wherein themobile phase modifier is an amine. The method as recited above, whereinthe amine mobile phase modifier is selected from the group consistingof: methylamine, ethylamine, diisopropylamine, diethylamine,dimethylamine, ethylmethylamine, triethylamine, propylamine, aniline anddimethylaniline. The method as recited above, wherein the mobile phasemodifier is an amino acid or amino acid ester. The method as recitedabove, wherein the amino acid or amino acid ester mobile phase modifieris selected from the group consisting of: L-amino acids, D-amino acids,L-amino acid esters and D-amino acid esters. The method as recitedabove, wherein the stationary phase is silica gel. The method as recitedabove, wherein the amino acid or amino acid ester mobile phase modifieris selected from: L-proline, D-proline, trans-4-hydroxy-L-proline,trans-4-hydroxy-D-proline, glycine, L-threonine, D-threonine, L-lysine,D-lysine, L-methionine, D-methionine, D-valine, L-valine and esters ofthe aforementioned b-and D-amino acids. The method as recited, whereinthe mobile phase is a solvent system comprising water, methanol, andethyl acetate. The method as recited above, wherein the amino acidmobile phase modifier is selected from: L-proline and D-proline.

Yet another embodiment of the invention is the method of purifying apeptide or a lipopeptide by using an amine or amino acid mobile phasemodifier in a normal phase chromatography system with a stationary phaseof alumina or silica gel to improve the resolution and/or productivityof the purification, wherein the purification is of a peptide. Themethod as recited above, wherein the mobile phase modifier is an amine.The method as recited above, wherein the mobile phase modifier is anamino acid or amino acid ester. The method as above, wherein thestationary phase is silica gel.

Examples of lipopeptides, for which this purification process is useful,are echinocandin derivatives, such as Pneumocandin B₀, Caspofungin,Cilofungin and Micafungin as well as Anidulafungin and Daptomycin, andparticularly the natural product precursors of Caspofungin, Micafungin,Cilofungin, Anidulafungin and Daptomycin. The naturalproduct/fermentation product precursor for Caspofungin is PneumocandinB₀. Caspofungin acetate (CANCIDAS) is a semisynthetic lipopeptideechinocandin B derivative currently being sold in the US as anantifungal agent for intravenous administration. Anidulafungin is asemisynthetic lipopeptide echinocandin B derivative under development byEli Lilly/Versicor as an antifungal agent for intravenousadministration. Anidulafungin is disclosed in U.S. Pat. Nos. 5,965,525and 6,384,013, hereby incorporated by reference. Cilofungin is anechinocandin lipopeptide disclosed by Eli Lilly in U.S. Pat. No.4,293,489 for use as an antifungal agent, hereby incorporated byreference. Micafungin (FUNGARD) is an echinocandin-like lipopeptideunder development by Fujisawa, as an antifungal agent for intravenousadministration. Micafungin is disclosed in U.S. Pat. No. 6,107,458hereby incorporated by reference. Daptomycin (CIDECIN) is asemisynthetic lipopeptide derivative under development by Cubist as anantibacterial agent. Daptomycin is disclosed by Eli Lilly in U.S. Pat.No. 4,537,717 hereby incorporated by reference.

A normal phase chromatography system employs a mobile phase and astationary phase. The mobile phase is a solvent system comprising one ormore solvents, the composition of which is either constant through outthe purification process, or a gradient, where the solvent compositionis changed over time during the purification process. The mobile phasesolvents include, but are not limited to, water, methanol, ethanol,isopropanol, hexane, ethyl acetate, acetonitrile, and methylenechloride. The stationary phase is selected from silica gel and alumina.The instant invention provides a chromatographic purification method fora peptide or lipopeptide, which employs the addition of a mobile phasemodifier to the mobile phase. A column volume is defined, as the volumeof solvent needed to traverse the column. Column load (also referred toas column feed or feed load) refers to the amount of material (crudelipopeptide or peptide) that can be purified at one time. The mobilephase modifier is defined as an amino acid, an amino acid ester, oramine. The instant invention contemplates the addition of the mobilephase modifier either to the eluant (mobile phase solvent system) or asa supplement to the column load. Examples of the amino acidscontemplated by the invention are any natural or unnatural amino acids,including both L- and D-configurations. Embodiments of the amino acidmobile phase modifiers useful in this process are L- and D-proline,trans-4-hydroxy-L-proline, glycine, L-threonine, L-lysine, L-methionineand L-valine. Examples of amine mobile phase modifiers contemplated bythe invention are primary, secondary, or tertiary lower alkyl (C₁-C₆alkyl) and/or aromatic (C₆-C₁₀ aryl) amines. Examples of such amines aremethylamine, ethylamine, diisopropylamine, diethylamine, dimethylamine,ethylmethylamine, triethylamine, propylamine, aniline anddimethylaniline.

This invention shows that adding low concentrations of certain amines,amino acids, or amino acid esters to the mobile phase can shift theresolution and selectivity of the system, providing significantenhancement. In particular, the addition of proline to theEtOAc-MeOH-water mixture used in the silica gel chromatography ofPneumocandin B₀ significantly increases the resolution of the compoundfrom closely related impurities, including Pneumocandins B₅ and E₀. Thisincreased resolution allows the loading of the product onto the columnto be significantly increased, by a factor of 2 to 3-fold, whilemaintaining the purity of the product rich cut and the yield. Largeramounts of product can be purified to the same purity at the same yieldon the same column of silica using less solvent and in less time. It hasalso been demonstrated that the presence of the mobile phase modifier inthe column load will have a similar effect.

Solvents purchased from Fisher Scientific (Pittsburgh, Pa., USA) wereused. Analytical scale HPLC columns (250×4.6 mm id) packed with regularbare silica, 5 μm particle size and 120 Å pore diameter, were obtainedfrom YMC (Wilmington, N.C., USA). The semi-preparative scale experimentemployed irregular silica from Amicon (Beverly, Mass., USA) designatedGrade 631, Si-60 of nominal particle size 20 μm. For analytical scaleexperiments, all solvents in the ternary mobile phase and the feeddiluent (ethyl acetate, methanol and water) were HPLC grade. Mobilephase employed for the semi-preparative experiment was prepared frommethanol (99.9% w/w purity) from Enron (Houston, Tex., USA) and urethanegrade ethyl acetate from Eastman (Perth Amboy, N.J., USA) and low ionwater produced in the laboratory using a purification unit from Osmonics(Minnetonka, Minn., USA). For both the analytical and semi-prep scaleexperiments, the mobile phase composition was 87/9/7 (v/v/v) ethylacetate/methanol/water. Volumetric compositions of solutions do notaccount for non-ideal mixing effects. For the sake of convenience, themobile phase will be referred to as WEAM (water/ethyl acetate/methanol).The feed used for the experiments contained Pneumocandin B₀ dissolved ina solvent blend of 79.3/13.3/7.4, v/v/v ethyl acetate/methanol/water.L-proline was obtained from Kyowa Hakko Kogyo Ltd. (Japan). Glycine,L-threonine, D-proline, L-methionine, L-lysine, L-valine,trans-4-hydroxy-L-proline, diethyl amine and proline methyl ester wereobtained from Sigma-Aldrich Cheme Gmbh (Steinheim, Germany).

Analytical scale experiments were performed on a Hewlett Packard(Waldbronn, Germany) HP1100 HPLC system with column thermostat andvariable wavelength detector (VWD) with detection monitored at 278 nm.Semi-preparative experiments employed a Dorr Oliver Model C (1 LPMpumping skid, Biotage Division of Dyax) and a 50 cm×6 cm id axialcompression column from Prochrom (Indianapolis, Ind.).

The modifier effect was first observed during the purification of aparticular batch of crude Pneumocandin B₀ product containing L-proline,which had been added into the fermentation mixture to facilitate thefermentation process.

When the crude fermentation product of Pneumocandin B₀ was first loadedto the HPLC, the resolution of Pneumocandin B₀ was limited. PneumocandinE₀ was buried under the peak for Pneumocandin B₀ and the separation ofPneumocandin B₅ from Pneumocandin B₀ was not prominent. See FIG. 1A,which represents the UV (278 nm) traces for the 1^(st) run of the silicagel HPLC.

However, after a few sequential HPLC purification runs of thisPneumocandin B₀ fermentation product, an increase in the retention timeof Pneumocandin B₀ by as much as 10 minutes was observed. In addition,Pneumocandin E₀, the Pneumocandin B₀ that co-eluted in earlier runs wasnow eluting ahead of the main peak. Furthermore, the resolution ofPneumocandin B₅, the close eluter of Pneumocandin B₀, improveddramatically. FIG. 1B presents the 18^(th) in this series of HPLC runsand clearly shows this increase in retention time and enhancedresolution.

The enhanced separation was reversed by a methanol wash of the column,which was performed every twenty runs for the purpose of removingtightly bound impurities. The improved purification results returnedagain after a few additional runs. Further investigation determined thatthe effect was due to the presence of proline in the-column feed (thecrude Pneumocandin B₀), which adsorbed to the column with eachinjection. This phenomenon was generalized by addition of proline andrelated additives to the mobile phase so that the level of the effectcould be controlled, providing improvements in resolution andproductivity, as described in the examples.

The examples provided herein are intended to assist in a furtherunderstanding of the invention. Particular materials, employed speciesand conditions are intended to be further illustrative of the inventionand not limitative of the reasonable scope thereof.

EXAMPLE 1

On an analytical scale YMC 5 μ silica column using 87/9/7 v/v/v ethylacetate/methanol/water as the mobile phase, a series of 8 injections ofPneumocandin B₀ product containing no L-proline were carried out toestablish an unchanging control for comparison. Following this, 10 shortinjections of an L-proline solution (1.3×10⁻²M L-proline in79.3/13.3/7.4 v/v/v ethyl acetate/methanol/water, 60 μL injection, about5 min run time at 1.2 mL/min) were made, and nine additional injectionsof the Pneumocandin B₀ product containing no L-proline were carried out.The procedure was repeated if necessary. FIG. 2A shows the retentiontime for the Pneumocandin Bo peak in the system for injections madeimmediately after the series of proline injections, relative to theretention time prior to proline injection. It can be seen that there wasa marked increase in retention immediately after L-proline treatment.This was accompanied by an improvement in the selectivity betweenPneumocandin B₀ and its immediate early eluting peak. However, theretention time returned to normal (RRT˜1) after about 5 runs and theimproved selectivity was lost, presumably due to the desorption ofL-proline with continued elution from the column with the proline-freemobile phase.

FIGS. 2B and 2C are typical chromatograms obtained from the injectionsjust before and just after the L-proline treatment. The change inretention is apparent, as is a distinct change in resolution betweenPneumocandin B₀ and Pneumocandins B₅ and E₀.

EXAMPLE 2

Experiments were carried out on a 5 Tm YMC silica-packed analyticalcolumn with L-proline added directly to the mobile phase. FIGS. 3A, Band C shows chromatograms without L-proline and with 0.26 mM (˜30 mg/L)and 0.65 mM (˜75 mg/L) L-proline in the mobile phase, respectively. Thecolumn was equilibrated for 2 hours at 1.2 ml/min with the prolinecontaining mobile phases before the injections were carried out. In FIG.3C, the peak just ahead of Pneumocandin B₀ was determined to bePneumocandin E₀ by the injection of standards Pneumocandin E₀ was notseparable when the chromatography was carried out without L-proline.

EXAMPLE 3

The selectivity enhancement was studied by increasing the retention timeof Pneumocandin B₀ to around 23 minutes, which is comparable to theretention time of around 22 minutes seen when 0.65 mM L-proline was usedin Example 2, by eluting with a mobile phase of 20% ethyl acetate and80% of the 87/9/7 v/v/v ethyl acetate/methanol/water. Although theretention time of Pneumocandin B₀ was increased, Pneumocandin E₀ wasstill buried in the peak of Pneumocandin B₀. No enhanced resolution wasachieved. See FIG. 4.

EXAMPLE 4

On a semi-preparative scale, a 6 cm ID Prochrom column was packed with390 g of Amicon Grade 631, 18-20 μ, 60 Å silica (slurried in a 87/9/7v/v/v water/ethyl acetate/methanol (WEAM) mobile phase; packing pressure40 bar; column length 25 cm) was used.

Benchmark Run:

A feed solution was prepared by dissolving 90 g of crude Pneumocandin B₀without L-proline in 1.5 L feed solvent (79.3/13.3/7.4 v/v/v ethylacetate/methanol/water) giving a 34.9 assay g/L Pneumocandin B₀ feedsolution. In order to establish a control for the experiment, 80 mL ofthe feed (2.8 g Pneumocandin B₀) were injected and eluted with the WEAMmobile phase at a flow rate of 90 mL/min. Fractions were collected fromthis run and analytical results of a pooled representative rich cutindicated that the impurity profile was consistent with previous batches(by both normal phase (NP) and reversed phase (RP) HPLC assays).

Runs with L-proline in the Mobile Phase(Similar Load to Benchmark):

The WEAM mobile phase was modified with L-proline at a concentration of75 mg/L (0.650 mM). The column was conditioned with 5 column volumes(CV) of this solution and a run was carried out with the modified mobilephase under conditions otherwise similar to the benchmark runs. Theshift in retention time observed in this system was not as dramatic asin the analytical system. Table 2 summarizes the impurity profiles ofthe rich cuts from the two runs: levels of Pneumocandin B₅ andPneumocandin E₀ are dramatically lower in the proline modified run,showing that higher throughputs could be achieved with this system.

TABLE 2 Summary of rich cut impurity profiles from laboratory runs,standard loading, without and with 0.15 g/L (1.3 M) of L-proline in themobile phase Load as grams Pneumocandin B₀ Pn B₅ Pn E₀ Yield Mass Runper kg silica % Area % Area % Balance % Benchmark 7.2 0.44 0.42 82 99run L-proline 7.2 0.16 0.05 84 98 systemDoubling Column Throughput with the L-proline Modified Mobile Phase:

A run was carried out using the proline-modified system described abovewith double the benchmark load: 5.6 g Pneumocandin B₀ injected (14.4g/kg silica) by doubling the volume of the feed solution injected, allother conditions being the same. The impurity profile of the rich cut,as well as the yield and mass balance, is summarized in Table 3. Theresults indicate the potential use of this method to double theproductivity of the chromatography step, since in this example the yieldhas been maintained while increasing purity at double the benchmarkloading.

TABLE 3 Summary of rich cut impurity profiles from laboratory runs, 2×loading, without and with 0.075 g/L (0.65 M) and 0.15 g/L (1.3 M)L-proline in the mobile phase Load: g Pneumocandin B₀ Pn B₅ Pn E₀ YieldMass Run per kg silica % Area % Area (%) Balance (%) Benchmark 7.2 0.440.42 82 99 L-proline 7.2 0.16 0.05 84 98 system L-proline 14.4 0.27 0.0886 99 system

EXAMPLE 6 Other Amino Acids as Mobile Phase Modifiers

In order to obtain further insight regarding the phenomenon, a series ofanalytical experiments were carried out using a 25×0.46 cm YMC 5 μsilica column with a wide variety of amino acid modifiers, includingtrans-4-hydroxy-L-proline, L-valine, L-lysine, L-methionine, D-proline,L-threonine and glycine in the mobile phase at a concentration of 0.65mM. See FIGS. 5-11.

Table 4 sorts the amino acids in order of decreasing retention. FIGS. 5through 10 provide exemplary chromatograms obtained from these runs thatillustrate their various effects on retention time and selectivity. Withevery amino acid except 4-hydroxy-L-proline, retention increasedcompared to the amino acid-free case, and the selectivity was modifieduniquely in each instance. For example, althoughtrans-4-hydroxy-L-proline did not cause the retention of Pneumocandin B₀to shift, it did affect the resolution of Pneumocandin B₀ from the earlyeluting peak, as seen in FIG. 5.

Glycine has no side chain, but it also increases the retention time.This strongly suggests that the retention time shift and the improvedresolution is not the unique effect of the amino acid side chain.

L-proline methyl ester was employed, in place of an amino acid and hadan increased retention time (retention time of Pneumocandin B₀ relativeto the control was 1.65) and a change in selectivity.

After each set of 3 or 4 runs with a particular amino acid, the columnwas washed with 36 ml (˜8 column volumes, hereinafter referred to as CV)of methanol and then re-equilibrated with 10 CV of neat WEAM mobilephase, after which three (˜30 min) injections were carried out toconfirm that the amino acid had been removed (i.e., that baselineperformance had been recovered). Following this, the column wasre-conditioned with 72 mL (˜17 CV) of the mobile phase containing a newamino acid, and new injections were then made. The retention timesreported are for the 3^(rd) run in each case. Since it is unclear whatthe saturation capacity of the silica is for the different amino acids,this protocol ensured that in each case the column had been exposed tothe same number of moles of the amino acid. The column could not beadequately regenerated after exposure to L-lysine (which was purposelytested as the last amino acid), presumably due to the two primaryamines.

TABLE 4 Retention times of Pneumocandin B₀ on a YMC 5μ, 120 Å, silicacolumn 25 cm × 0.46 cm ID at a flow rate of 1.2 mL/min. Amino Acid (65mM) Retention of Pneumocandin B₀ in WEAM relative to the controlL-proline methyl ester 1.65 L-proline 1.44 Glycine 1.38 L-threonine 1.35D-proline 1.25 L-methionine 1.16 L-lysine 1.13 L-valine 1.10trans-4-hydroxy-L-proline 1.00 None (control) 1.00

EXAMPLE 7 Chromatography with Diethylamine as a Mobile Phase Modifier

Diethylamine was added to the mobile phase at about 0.1 g/L, and theexperimental conditions were identical to that employed previously inExample 6. As shown in FIG. 12, the retention time increased even morewith diethylamine than with proline. Therefore, the influence onretention and selectivity appears to be mediated by the aminefunctionality, such that either an amine or an amino acid/amino acidester could be used as a mobile phase modifier in this system.

1. A method of purifying a peptide or a lipopeptide by using a mobilephase modifier in a normal phase chromatography system to improve theselectivity and/or productivity of the purification, wherein the mobilephase modifier is selected from a group consisting of an amino acid andan amino acid ester, the normal phase chromatography system includes amobile phase and a stationary phase, the mobile phase is a solventsystem comprising one or more solvents, and the stationary phase isselected from silica gel and alumina, except that when the lipopeptideis Pneumocandin B₀, then the mobile phase modifier is not L-proline. 2.The method as recited in claim 1, wherein the amino acid or amino acidester mobile phase modifier is selected from the group consisting of:L-amino acids, D-amino acids, L-amino acid esters and D-amino acidesters.
 3. The method as recited in claim 2, wherein the amino acid oramino acid ester mobile phase modifier is selected from: L-proline,D-proline, trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline,glycine, L-threonine, D-threonine, L-lysine, D-lysine, L-methionine,D-methionine, D-valine, L-valine and esters of the aforementioned L-andD-amino acids.
 4. The method as recited in claim 3, wherein the aminoacid is selected from: L-proline and D-proline.
 5. The method as recitedin claim 1, wherein the normal phase chromatography system is for thepurification of a peptide.
 6. The method as recited in claim 5, whereinthe peptide is oxytocin or bradykinin.
 7. The method as recited in claim1, wherein the normal phase chromatography system is for thepurification of a lipopeptide.
 8. The method as recited in claim 7,wherein the lipopeptide is a fermentation product precursor ofcaspofungin, micafungin, cilofungin, andulifungin and daptomycin.
 9. Themethod as recited in claim 8, wherein the fermentation product precursorof caspofungin is pneumocandin B₀.
 10. The method as recited in claim 9,wherein the amino acid or amino acid ester mobile phase modifier isselected from the group consisting of: L-amino acids, D-amino acids,L-amino acid esters and D-amino acid esters, except the L-amino acid isnot L-proline.
 11. The method as recited in claim 10, wherein the aminoacid or amino acid ester mobile phase modifier is selected from:D-proline, trans-4-hydroxy-L-proline, trans-4-hydroxy-D-proline,glycine, L-threonine, D-threonine, L-lysine, D-lysine, L-methionine,D-methionine, D-valine, L-valine and esters of the aforementioned L-andD-amino acids.
 12. The method as recited in claim 11, wherein the aminoacid mobile phase modifier is D-proline.
 13. The method as recited inclaim 1, wherein the mobile phase is a solvent system comprising water,methanol, and ethyl acetate.
 14. The method as in claim 1, wherein thestationary phase is silica gel.
 15. A method of purifying pneumocandinB₀ by using a mobile phase modifier in a normal phase chromatographysystem to improve the selectivity and/or productivity of thepurification, wherein the normal phase chromatography system includes amobile phase and a stationary phase, the mobile phase is a solventsystem comprising one or more solvents, the stationary phase is selectedfrom silica gel and alumina, and the mobile phase modifier is selectedfrom the group consisting of: methylamine, ethylamine, diisopropylamine,diethylamine, dimethylamine, ethylmethylamine, triethylamine,propylamine, aniline and dimethylaniline.
 16. The method as in claim 15,wherein the stationary phase is silica gel.
 17. A method of purifyingoxytocin or bradykinin by using a mobile phase modifier in a normalphase chromatography system to improve the selectivity and/orproductivity of the purification, wherein the normal phasechromatography system includes a mobile phase and a stationary phase,the mobile phase is a solvent system comprising one or more solvents,the stationary phase is selected from silica gel and alumina, and themobile phase modifier is selected from the group consisting of:methylamine, ethylamine, diisopropylamine, diethylamine, dimethylamine,ethylmethylamine, triethylamine, propylamine, aniline anddimethylaniline.